DE4403666A1 - Subunits of glutamate receptors, processes for their preparation and their use - Google Patents

Abstract

The invention pertains to new glutamate receptor subunits and DNA sequences coding for them and to methods for producing DNA sequences and receptors. The invention also pertains to methods for identifying functional ligands for these receptors.

Description

The invention relates to the expression of new ionotropic variants Glutamate receptor subunits in eukaryotic cells and Ver drive to find functional ligands for corresponding Glutamate receptor channels.

Glutamate is the most important excitatory neurotransmitter in the central nervous system (TIPS 11, 1990, 126-132; Pharmacological Reviews 40, 1989, 143-210; TIPS 13, 1992, 291-296) and is in numerous pathophysiological processes such as B. epilepsy, Schizophrenia, ischemia involved. Are receptors for glutamate therefore potential targets for corresponding pharmaceuticals.

Some subunits have so far been elucidated in their primary structure units of AMPA, Kainat and NMDA receptors as well as some meta botrope receptors (Nature 342, 1989, 643; Science 249, 1990, 556; Neuron 8, 1992, 169; Science 256, 1992, 1217; Nature 358, 1992, 36).

So far, four AMPA glutamate receptor sub have been found in the literature units of the rat described, GluRA, GluRB, GluRC and GluRD, each in two splicing variants, "flip" and "flop" come (Science 249, 1990, 1580). For mouse and rat GluRB "RNA editing" has also been shown, the so-called Q / R Site of the second transmembrane domain. These two GluRB variants differ considerably in their electrophy Siological properties (Cell 67, 1991, 11-19; Neuron 8; 1992, 189-198). The human cDNA for GluRAflip and GluRAflop is flat if published (PNAS USA 88, 1991, 7557-7561; PNAS USA 89, 1992, 1443-1447).

There have now been variants of the human glutamate receptor subunit ten A, B, C and D found, as well as DNA sequences for such Encode subunits. These subunits lead to GluR Channels with specific electrophysiological properties.

For GluRA, GluRB, GluRC and GluRD it was found that the first Amino acid of the "flip / flop" region by "RNA editing" as Glycine (G) or as arginine (R) can be present. The correspond the subunits are identified as follows:

GluRAflipG, GluRAflipR, GluRAflopG, GluRAflopR
GluRBflipQ-G, GluRBflipQ-R, GluRBflopQ-G, GluRBflopQ-R
GluRBflipR-G, GluRBflipR-R, GluRBflopR-G, GluRBflopR-R
GluRCflipG, GluRCflipR, GluRCflopG, GluRCflopR
GluRDflipG, GluRDflipR, GluRDflopG, GluRDflopR.

At GluRB, the "RNA editing" already known to the rat taken into account when designating the corresponding variants been.

For GluRA, an "alternative splicing "variant found, in which a 240 bp frag The 5'-region of the GluRA cDNA is missing and the corresponding Protein is thus shortened by 80 amino acids. The corresponding Subunits are identified as follows:

GluRAdel240flipG, GluRAdel240flipR, GluRAdel240flopG, GluRAdel240flopR.

The following DNA or amino acid sequences refer to finally on human glutamate receptor subunits.

In SEQ ID NO: 1 is the cDNA sequence of GluRAflipG and that thereof derived polypeptide sequence (SEQ ID NO: 2) shown; in SEQ ID NO: 3 the cDNA sequence of GluRAflopG, in SEQ ID NO: 4 that thereof derived polypeptide sequence.

The GluRAflipR cDNA has cDNA compared to the GluRAflipG a base exchange at position bp 2269, which is a glycine codon (GGA) converted to an arginine codon (AGA). The following applies to GluRAflopR corresponding.

The GluRAdel240 variants correspond to the GluRA Variants, however, have a deletion: bp 221-460 based on SEQ ID NO: 1 and 3.

In SEQ ID NO: 5 is the cDNA sequence of GluRBflipQ-G and the polypeptide sequence derived therefrom (SEQ ID NO: 6) is shown; in SEQ ID NO: 7 the cDNA sequence of GluRBflopQ-G, in SEQ ID NO: 8 the derived polypeptide sequence.

The cDNA for GluRBflipQ-R has compared to GluRBflipQ-G a base exchange at position bp 2290, which is a glycine codon (GGA) converted to an arginine codon (AGA). The following applies to GluRBflopQ-R corresponding.  

The cDNA molecules for GluRBflipR-G, GluRBflipR-R, GluRBflopR-G and GluRBflopR-R have compared to the above GluRB Varian base exchange at position bp 1820, which is a glutamine Converts codon (CAG) into an arginine codon (CGG).

In SEQ ID NO: 9 is the cDNA sequence of GluRCflipG and in SEQ ID NO: 10 the polypeptide sequence derived therefrom. SEQ ID NO: 11 shows the cDNA sequence of GluRCflopG and SEQ ID NO: 12 the derived polypeptide sequence.

The cDNA molecules for GluRCflipR and GluRCflopR have in Ver same as GluRCflipG or GluRCflopG a base exchange at Po sition bp 2377, which converts a glycine codon (GGA) into an arginine codon (AGA) converts.

In SEQ ID NO: 13 is the cDNA sequence of GluRDflipG and in SEQ ID NO: 14 shows the polypeptide sequence derived therefrom. SEQ ID NO: 15 shows the cDNA sequence of GluRDflopG and SEQ ID NO: 16 the derived polypeptide sequence.

The cDNA molecules for GluRDflipR and GluRDflopR have in Ver a base exchange at the bottom of GluRDflipG or GluRDflopG sition bp 2293, which converts a glycine codon (GGA) into an arginine codon (AGA) converts.

Other suitable DNA sequences are those that are different Nucleotide sequence as that in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or Have 15 listed, but due to the degeneration of genetic codes for those in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or encode 16 listed polypeptide chain or parts thereof. DNA sequences suitable for AMPA-Glut are also suitable encode amate receptor subunits and those under standard conditions conditions with the in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 Darge put nucleotide sequence or with a nucleotide sequence that for that shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 Protein encodes hybridize. Under standard conditions are for example temperatures between 42 and 58 ° C in an aqueous Buffer solution with a concentration between 0.1 and 1 × SSC (1 × SSC: 0.15M NaCl, 15mM sodium nitrate pH 7.2). The experimental conditions for DNA hybridization are in Textbooks on genetic engineering, for example in Sambrook et al., "Molecular Cloning," Cold Spring Harbor Laboratory, 1989, be wrote.

Furthermore, genetic engineering manufacturing processes for these sub units found. It was also found that the for DNA sequences encoding these receptor subunits  find functional ligands to use for these receptors to let. The invention also relates to methods to identify functional ligands for AMPA glutamate receptors, which are characterized in that sequences, which code for AMPA-GluR subunits, transfi cells adorned, the membranes of these cells isolated and with this mem conducts industry-standard receptor binding experiments.

Another identification method according to the invention This makes functional ligands for AMPA glutamate receptors characterized in that cells with one or more DNA sequences were transfected for AMPA-GluR subunit encode, influencing the signal transduction path caused by binding of the ligands to the receptor by a receptor system is detected, for example the intracel Lular Ca⁺⁺ concentration after ligand binding with fluorimetri methods (Anal. Biochem. 209, 1993, 343).

The new polypeptides and DNA sequences can be genetically engineered using known methods. So you can look out Isolate brain tissue mRNA and translate it into double-stranded cDNA. This cDNA can serve as a template for the polymerase chain reaction be used. By using specific primers the appropriate cDNA under suitable reaction conditions be amplified. By using suitable primers the amplified cDNA sequenced without prior cloning become. The double-stranded cDNA can also be used in λ vectors, e.g. B. λ gt 10 or λ ZAP, can be integrated to a brain-specific generate cDNA bank. Such a cDNA bank can be used with radioactive tively labeled DNA or RNA probes are searched for clones identify the homology with the hybridization sample exhibit. The methods used are, for example, in "Current Protocols in Molecular Biology" (Ed. F.M. Ausubel et al.) 1989, ISBN 0-471 50338-x (Vol. 1 and 2), for the polymerase Chain reaction in Saiki et al. (1985) Science, 230, 1350-54 and Mullis and Faloona (1987) Meth. Enzymol., 155, 335-350 ben.

The cDNA characterized in this way is by means of restriction enzyme easily accessible. The resulting fragments, possibly in Connection with chemically synthesized oligonucleotides, Adapto genes or gene fragments can be used to identify the genome To clone protein coding sequences. The installation of the gene fragments or synthetic DNA sequences in cloning vector ren, e.g. B. the commercially available plasmids M13mp18 or Bluescript, takes place in a known manner. Also the genes or gene question elements with suitable chemically synthesized or from bacteria,  Control phages, eukaryotic cells or their viruses isolated regions are provided which the expression of the proteins in enable different host systems.

The transformation or transfection of suitable host organisms with hybrid plasmids is also known and detailed (M. Wigler et al., Cell, 16 (1979), 777-785; F.L. Graham and A.J. van der Eb, Virology, 52 (1973), 456-467).

Vectors can be used for expression in mammalian cells, which is the gene to be expressed, in this case the one for here described subunits of AMPA glutamate receptors codie cDNA sequences under the control of the mouse metallothio no, the SV40 viral or the cytomegalovirus promoter set (J. Page Martin, Gene, 37 (1985), 139-144). Necessary the expression is the presence of the methionine start codon of the gene responsible for these subunits of AMPA glutamate receptor encoded. Clones are then isolated, copies of these vectors have as episomes or integrated into the genome. Especially before the integration of the foreign gene into a vector is partial contains the promoter of the cytomegalovirus.

Alternatively, one can use a suitable vector of cells art transfect that the transient expression of the so brought DNA for a pharmacological characterization of the ex primed heterologous polypeptides is sufficient. Here too is the Control of expression by the cytomegalovirus promoter particularly advantageous.

It can also be used to produce functional AMPA glutamate receptors produce that one or more different DNA sequences from the group of the AMPA-GluR subunits together in cells transfected. In this way, AMPA glutamate receptors can be win with different subunits.

In connection with prokaryotic sequences which are necessary for the Repli cation in bacterial cells and an antibiotic resistance code ren, the use of "shuttle" vectors is well suited. Con The plasmid is first constructed and propagated in Bak serial cells; the conversion into the eukaryon then takes place cells, e.g. B. in the human embryonic kidney cell line HEK 293.  

Other cell systems, e.g. B. yeast and other fungi, insect cell len as well as animal and human cells such. B. CHO-, COS- and L- Cells can be associated with appropriate expression vectors can be used to express the cloned cDNA.

The eukaryotic expression systems have the advantage that they are able to use their products effectively and mostly in native Express form. They also have the ability to pro to modify products post-translationally.

The expressed receptor proteins can be solu by detergents be bilized and known by affinity chromatography procedures. The pure polypeptide can, according to Crystallization and X-ray structural analysis or other physika processes such as NMR or scanning tunneling microscopy are used, first the spatial structure of the receptor and then the spatial structure of the ligand binding site ren.

The expressed receptor proteins can after appropriate Rei also as antigens for the generation of polyclonal or serve monoclonal antibodies. These antibodies in turn can may be used for diagnostic purposes. A Another application for such antibodies is in their use as tools for rational drug design. So can use the receptor-specific antibodies as an antigen for the Generation of anti-idiotypic antibodies can be used. Sol Che antibodies can be an image of the Re represent zeptors and for screening for specific recipes goal ligands or for rational drug design.

Receptor-expressing cell lines are an important tool in the screening for specific receptor ligands the membranes of these cells are used for receptor binding tests become. Information on mode of action (agonism / antagonis mus) of a receptor ligand can be obtained in that cells transfi with a DNA sequence according to the invention have been decorated with a suitable reporter system. Suitable reporter systems are those in which a promoter, by connecting the signal transduction path (second mes senger) is regulated with a gene for an easy to detect product such as luciferase is functionally linked. Such Reporter systems are for example from Science 252, 1424 (1991); Proc. Natl. Acad. Sci. USA 88, 5061 (1991) or Journal of Recep tor Res. 13, 1993, 79. A suitable promoter that is used in for example regulated by the intracellular Ca⁺⁺ concentration is that of the fos gene. It is also possible to change the  intracellular Ca⁺⁺ concentration with fluorescent dyes (e.g. FURA 2AM) directly.

Furthermore, the current flow through the cell membrane can be dependent of ligand binding can be measured.

Due to the degeneration of the genetic code, it is possible other DNA sequences than described here, e.g. B. chemically synthe genes with different DNA sequences for the express sion of the described subunits of human AMPA glutamate to use receptors.

With the help of the invention it becomes possible to identify substances grace and characterize the Re bind the receptor and act there agonistically or antagonistically.

Another object of the invention is the use of oli gonucleotides derived from that shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 structure are derived as antisense Mo lenses for the targeted elimination of genes.

With the help of the invention it is also possible to use synthetic oligo to produce nucleotides with which the expression of AMPA Glutamate receptor subunits through intracerebroventricular Application can be specifically inhibited, such as has been described for NMDA receptors (Nature 363, 1993, 260).

Further refinements of the invention are given in the examples described here.

For genetic engineering methods such. B. on the manual of Sambrook et al. "Molecular Cloning", Cold Spring Harbor Labora tory, 1989, or "DNA cloning", vol. I to III, IRI Press 1985 until 1987, publisher D.M. Glover, pointed out.

example 1

Isolation of cDNA molecules required for human AMPA glutamate encode receptor subunits GluRA and GluRAdel240.

Using the technique of polymreas chain reaction (PCR), three cDNA fragments required for the human AMPA glutamate receptor sub unit GluRA are specific, from two commercially available human brain cDNA libraries amplified. To increase the Specificity of the amplification was that described below 2-step PCR procedure carried out: 10 µl lysate each  human temporal cortex cDNA library (titer: 1.5 x 1011 Phage / ml; Vector lambda ZAP; GluRA cDNA fragment bp 1-839) or a human nucleus accumbens cDNA library (titer: 1-9 × 10⁹ Phage / ml; Vector lambda gt10; GluRA cDNA fragments 1-1421 and 1407-2721) were used as a template for the first PCR reaction with the Primer oligonucleotides A and B used. The reaction volumes each of the batches was 100 μl, each 20 pmol of the various which primers were used and the reaction buffer contained 10 mM Tris-HCl pH 8.5, 50 mM KCl, 1.5 mM MgCl₂, each 0.2 mM dATP, dCTP, dGTP and dTTP. There were 20 cycles with the following Temperature profile performed: 1 minute 94 ° C, 1 minute 55 ° C, 1 minute 72 ° C. A type 9600 thermal cycler was used Perkin-Elmer company.

After the PCR reaction was carried out, 10 .mu.l each PCR approaches taken and used as a template for a second PCR reac tion used with primers C and D. The reaction and puf Conditions were the same as for the first PCR However, the number of cycles has been increased to 35. The amplified cDNA fragments were made according to the manufacturer's instructions Use of the TA Cloning Kit from Invitrogen in the vector pCRII cloned.

The primer oligonucleotides for the amplification of human GluRA cDNA fragment spanning base pairs 1-1431 the following sequences:

The primer oligonucleotides for the amplification of human GluRA cDNA fragment spanning base pairs 1407-2721 the following sequences:

The primer oligonucleotides for the amplification of human GluRA cDNA fragments spanning base pairs 1-839 had the following sequences:

With the help of standard genetic engineering methods (see e.g. Sambrook et al. (1989) "Molecular Cloning", Cold Spring Harbor Laboratory) the amplified cDNA fragments were each full permanent coding regions of GluRA and GluRAdel240 composed.

Example 2 Isolation of cDNA Molecules for Human Glutamate Receptor Subunits A, B, C and D

cDNA fragments specific for human glutamate receptor subunits A, B, C and D were obtained by "screening" the following commercially available human brain cDNA libraries:
Hippocampus (Stratagene)
Cerebellum (Clontech and Stratagene)
Nucleus accumbens (Clontech).

PCR fragments of sizes 600-3000 bp were used as screening samples used by the cDNA molecules cloned in pBluescript were amplified for rat GluRA, B, C and D. It 1 ng plasmid DNA was used as a template. The Pri mer concentrations and buffer conditions for the PCR reactions corresponded to those in Example 1, but in each case 2 .mu.l of the dNTP labeling mixture from the DNA Labeling and Detection Kit from Boehringer Mannheim as the nucleotide source. It 35 cycles were carried out with the following temperature profile: 2 minutes 94 ° C, 2 minutes 55 ° C, 3 minutes 72 ° C. The Dig-dUTP mar cated fragments were made on a Seaplaque agarose gel nigt. The screening procedure was carried out according to the  Manual regulations for the above Kit performed. The GluR cDNA fragments of the lambda resulting from the screening Clones were found using the usual genetic engineering methods the cloned pBluescript vector and the complete cDNA- Composed of molecules of the different GluR variants.

Example 3 Transient expression of the cloned human GluR genes in HEK293 cells

Unless otherwise described, the Lindl und Bauer, "Cell and Tissue Culture", Gustav Fischer Verlag, carried out.

The GluR cDNA molecules from Examples 1 and 2 were included in Rah their isolation in conventional plasmids such. B. pBluescript (Stratagene) and pCRII (Invitrogen) cloned. The toilet nated GluR fragments each encompass the entire open Reading frame, including start and stop codon, and at least 40 bp of the 5'-non-translated preceding the start codon Regions.

For transient expression in eukaryotic cell lines the cloned GluR fragments into the expression vector pcDNA3 (Invitrogen) cloned. The resulting recombinant th plasmids were propagated in a known manner.

HEK 293 cells were cultured under standard conditions. To The cells were trypsinized in DMEM medium (Gibco) 3.7 g / l NaHCO₃ contained, recorded and 1.5 × 10⁶ cells / 10 cm Seeded petri dishes. The cells were then kept at 37 ° C. for 24 h and 5% CO₂ cultivated.

The DNA to be transfected was prepared as follows: 20 µg of the DNA solution (1 mg / ml), cleaned with the Quiagen® system, Diagen, were mixed with 437 ul H₂O, then 62.5 ul 2 M CaCl₂ added and finally 500 ul BBS. Within Ca⁺⁺ precipitates were formed at room temperature for 10 min.

The solution was placed on a 10 cm cell culture dish with the following given above cultured HEK 293 cells. According to before The cells were mixed visually for 15 to 20 h in one Incubator cultivated at 37 ° C / 3% CO₂. After that, were careful 5 ml of serum-free medium was added. After removing the whole Medium and repeat washing with 5 ml medium 10 ml of medium were added to the cells. After 48 h incubation at 37 ° C  and 5% CO₂, the cells for pharmacological and electro physiological tests can be used.

Alternatively, the DNA was liposome-mediated into the cells brought in. Lipofectin from GIBCO-BRL Manufacturer information used accordingly.

Example 4 Expression of the AMPA glutamate receptor subunits in oocytes

The corresponding cDNA molecules, which code for the glutamate receptor subunits into which plasmid Bluescript (Stratagene) cleaved with EcoRI according to Stan cloned dard protocols.

After multiplying the Bluescript clones, which are for subunits of the AMPA glutamate receptors was encoded according to standard metho obtained the plasmid DNA. This plasmid DNA was labeled with the restriction tion enzyme Not I cleaved and used for in vitro transcription puts. The transcription was from the T3 or T7 promoter starts and under standard conditions according to the in vitro Stratagene transcription kit performed.

To express the receptor subunits, 10 ng each cRNA either individually or in combination with other cRNA in Oo injected with cytocytes from the clawed frog Xenopus laevis explan had been [C. Methfessel et al., Pflügers Arch. 407, 577, (1986)]. The oocytes were placed in OR-2 (92.5 mM NaCl, 2.5 mM KCl, 1mM Na₂HPO₄, 5mM HEPES, 1mM MgCl₂, 1mM CaCl₂, 0.5 g / l Po lyvinylpyrolidone, pH 7.2 with the addition of 4 µg / ml Zinacef and 100 U / ml Penstrep) incubated at 19 ° C. 24 hours after the injection tion, the oocytes were treated with collagenase (type II sigma) (1 mg / ml in OR-2 for 1 hour). Electrophysiological recordings were done 2-6 days after injection of the cRNA. Here a 2 electrode "voltage clamp" configuration was used with a TEC 01C amplifier (NPI Electronic, Tamm, Germany). Wuh During the electrophysiological measurements, the oocytes were analyzed normal Frog Ringer's solution NFR (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 10 mM HEPES, pH 7.2) perfused.  

Example 5 Stable expression of the glutamate receptor subunits in HEK 293 Cells

The glutamate receptor described in Examples 1 and 2 cDNA molecules were found in the eukaryotic expression vectors pcDNA3 and pRc / CMV (Invitrogen) cloned. These expressions Constructs were made by electroporation according to the following protocol introduced individually or in combination in HEK 293 cells: HEK 293 cells (ATCC) were in RPMI 1640 medium (Glutamax I, Gibco BRL) cultivated with 10% FCS (Gibco BRL) under 5% CO₂. For the electroporation 10⁷ cells in 0.8 ml PBS with 20 µg the expression construct with an electroporator (BTX, electro cell manipulator 600, 3 uF, 130 V, 72 ohms) transfected. The cell len were then incubated for 24 h in culture medium and on finally in selection medium (RPMI medium with 600 µg / ml G418 sulfate, geneticin) transferred. Stable geneticin resistant Cell clones were isolated after 10-12 days via single cell deposition, expanded and analyzed using a membrane binding assay.  

Sequence listing

Claims (7)

1. DNA sequences that code for variants of glutamate receptor subunits and those from the group that of

• a) DNA sequences of the structure described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15,
• b) DNA sequences which code for proteins with the structure described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, and
• c) DNA sequences which hybridize under standard conditions with DNA sequences a) or b) are formed,

are selected. 2. Process for the genetic engineering of glutamate receptor subunits using DNA sequences according to claim 1. 3. Use of DNA sequences according to claim 1 for identification decoration of functional ligands for glutamate receptors. 4. Methods for identifying functional ligands for Glutamate receptors, characterized in that with a for a DNA sequence coding for a glutamate receptor Claim 1 transfected cells, the membranes of these cells isolated and with these membranes usual receptor binding carries out experiments. 5. Methods for identifying functional ligands for Glutamate receptors, characterized in that with a or more DNA Se coding for a glutamate receptor Sequence according to claim 1 cells transfected and in these Cells caused by binding of the ligands to the receptor Influencing of the signal transduction path by a reporter system detected. 6. Use of oligonucleotides that differ from that in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 derived structure are, as antisense molecules for the targeted switching off of Genes.